Synthesis of gramine salt of nitroacetate esters

ABSTRACT

Tryptophan, an essential amino acid, is prepared from gramine and an ester of nitroacetic acid by combining gramine and the ester in an inert solvent maintained at about room temperature to form the corresponding salt, and then heating the salt in solution to convert it to the corresponding ester of Alpha nitro- Beta - (3-indole) propionic acid, which is reduced and hydrolyzed in accordance with conventional methods to afford tryptophan. The salt is a novel compound.

United States Patent Theodore Largman Inventor Morrlstown, NJ. Appl. No.832,832 Filed June 12, 1969 Patented Dec. 14, 1971 Assignee AlliedChemical Corporation New York, N.Y.

SYNTHESIS OF GRAMINE SALT OF [56] References Cited UNITED STATES PATENTS2,557,04l 6/1951 Weisblat et al. 260/326. 1 4 X Primary Examiner-AlexMazel Assistant Examiner-Joseph A. Narcavage AnorneysArthur J.Plantamura and Stanley M. Teigland ABSTRACT: Tryptophan, an essentialamino acid, is prepared from gramine and an ester of nitroacetic acid bycombining gramine and the ester in an inert solvent maintained at aboutroom temperature to form the corresponding salt, and then heating thesalt in solution to convert it to the corresponding ester of a-nitro-B-(3-indole) propionic acid, which is reduced and hydrolyzed in accordancewith conventional methods to afford tryptophan. The salt is a novelcompound.

SYNTHESIS OF GRAMINE SALT OF NITROACETATE ESTERS BACKGROUND OF THEINVENTION the body. Hence, they must be supplied in the diet. A lack of7 these amino acids in the diet results in malnutrition, and it has beenestimated that the diet of over half the world's population is lackingin these compounds. However, these amino acids can be synthesizedchemically and used to overcome nutritional deficiency, but this use hasbeen limited because of the high cost of synthesizing these compounds.Hence, it is desirable to develop improved methods of synthesizing theseessential amino acids in order to reduce their cost.

One of these eight essential amino acids is tryptophan. A method forproducing tryptophan is described in Lyttle and Weisblat, J. Am. Chem.Soc. 69,2118 (1947) and in U.S. Pat. No. 2,557,041, to Lyttle andWeisblat. The method described in these two references involves reactinggramine with an ester of nitroacetic acid to obtain the correspondingester of anitro-fi-(3-indole) propionic acid, which is catalyticallyreduced and hydrolyzed to afford tryptophan at a yield of about 45percent based on gramine. This method of synthesizing tryptophan isgenerally regarded as one of the most favorable routes to tryptophan.

SUMMARY OF THE INVENTION This invention provides a process for preparingthe gramine salt of an ester of nitroacetic acid, which represents aclass of novel compounds useful as intermediates in the preparation oftryptophan.

Lyttle and Weisblat teach that when gramine and an ester of nitroaceticacid are combined in an inert solvent and the temperature is raised toabout 90 C., the product obtained is the corresponding ester ofa-nitro-B-(S-indole) propionic acid. I have also found that if thetemperature is not raised, but rather is allowed to remain at about roomtemperature, the salt of gramine and the ester is formed and can bereadily recovered at a yield of 100 percent. The reaction leading to theformation of the salt is illustrated by the following equation:

CHzN(CHa)2 OINCHZCOOR The ester group, designated R, does notparticipate in the reaction and hence is not critical. Good results areobtained where R is lower alkyl, phenyl or lower alkyl substitutedphenyl.

The salt is obtained by adding gramine and an ester of nitroacetic acid,preferably in substantially equimolar quantities, to an inert solvent atabout room temperature, allowing the salt to precipitate and recoveringthe salt from the solvent by conventional methods, such as byfiltration, centrifugation, etc.

The discovery that gramine and an ester of nitroacetic acid combine toform a salt is surprising in and of itself, and is especially surprisingin view of the Lyttle and Weisblat references which do not appreciatethis phenomenon and which suggest. by negative inference, that a saltwould not be formed.

In order to form the salt, it is critical that the temperature of thesolution be maintained at less than about 40 C. At temperatures aboveC., the reactants combine to form the corresponding ester of a-nitro-B-(3-indole) propionic acid, as disclosed in the Lyttle and Weisblatreferences. Temperatures lower than room temperature can be used, butthere is no advantage in doing so.

The salt is useful as an intermediate in the preparation of tryptophan.When the salt is heated in an inert solvent at from about 90 to about l15 C., the corresponding ester of a-nitro- B-(3indole) propionic acid isformed at a yield which approaches percent. At temperatures below about90 C., the reaction is very slow, and at temperatures above about ll5 C.undesirable side reactions occur. Conversion of the salt to thecorresponding ester of a-nitro-B-(S-indole) propionic acid isillustrated by the following equation:

CHAJHCOOR system without interim recovery, to afford tryptophan, whichcan thus be prepared in a continuous process from the salt. Using thisprocess, tryptophan can be recovered at a yield of up to 80 percent ormore based on gramine. The reduction and hydrolysis steps are performedaccording to methods known in the art, e.g., as disclosed in the Lyttleand Weisblat references previously referred to herein.

In a third reference, J. Am. Chem. Soc., 71, 3079 (l949), Lyttle andWeisblat indicate that when the a-nitro-B- (3-indole) propionate esteris prepared directly from gramine and a nitroacetate ester, dialkylationof the nitroacetate ester by gramine is an undesirable side reactionwhich reduces the yield of the a-nitro-B-(S-indole) propionate ester andresults in a less pure product. For this reason, Lyttle and Weisblatappear to prefer to use a nitromalonate ester instead of a nitroacetateester even though the use of the nitromalonate ester requires anadditional step, viz, decarboxylation, in the overall process ofpreparing tryptophan. Furthermore, esters of nitroacetic acid areobtainable at a lower cost per mol than esters of nitromalonic acid. Inpreparing a-nitro-B-(3-indole) propionate esters from the salt ofgramine and an ester of nitroacetic acid, dialkylation is substantiallyavoided, thus permitting the use of the more preferred nitroacetateesters. Dialkylation is avoided because the two ions which comprise thesalt are associated in solution as a result of ionic attraction and areassociated in a one-to-one relationship. Hence, as dimethylamine isdriven off by heat, a nitroacetate group is immediately present tocombine with the 3-methylindole residue. Dialkylation does not occurbecause in order for it to occur the a-nitro-B-(3indole) propionatemolecule would have to attract a gramine cation away from a nitroacetateanion, which is not likely to occur.

in addition to avoiding dialkylation, another advantage of preparing thesalt as an intermediate is that it enables one to purify the initialreactants and combine them in a precisely one-to-one molar ratio.Nitroacetate esters are not available commercially. They are normallyprepared by treating nitromethane with concentrated potassium hydroxideand then esterifying the resulting dipotassium salt of nitroacetic acid.The esterified product is normally obtained as a residue with anappreciable amount of impurities. The nitroacetate ester can be readilyseparated from these impurities by dissolving the residue in an inertsolvent, adding gramine and precipitating the salt of gramine and thenitroacetate ester, which can be recovered and purified by simplewashing or reprecipitation. Hence, it is preferred, in an overallprocess of preparing tryptophan, to recover or isolate the salt prior toconverting it to the a-nitro-B-(Ii-indole) propionate ester.

The inert solvent employed in preparing the salt and theanitro-B-(S-indole) propionate ester is preferably nonpolar and includesstraight-chain and cyclic saturated hydrocarbons, and ethers. Toluene istypical and gives good results.

The invention is further illustrated by the following example.

EXAMPLE Preparation of Ethyl Nitroacetate In a 2-liter flask was placed1,410 grams of 15.9 percent solution of potassium hydroxide inn-butanol. Nitrogen was passed through the system as nitromethane (62grams, 1.0 mole) was added dropwise with vigorous stirring over a 50-minute period. The temperature rose from 25 to 50 C. during theaddition. The mixture was then refluxed overnight. After cooling, then-butanol was removed by decantation and the remaining solids werepurified by warming to 80 C. with 800 grams of 50 percent potassiumhydroxide in n-butanol. The mixture was cooled, filtered, and the cakewas washed with methanol. 63.3 grams of dry salt were recovered. Theprocedure was repeated and an additional 66.9 grams of salt wererecovered. I

Ethanol (1,000 ml.) was saturated with HC] (160 grams) and cooled to C.Over a 50-minute period, 129.5 grams of the dipotassium salt ofnitroacetic acid was added to the solution while maintaining thetemperature at -10 to -1 2 C. Stirring at 10 C. was continued for 2additional hours, after which the mixture was placed in a freezerovernight. The reaction was completed by a final stirring for 3 hours atC., followed by filtration to remove potassium chloride. The filtratewas diluted with an equal volume of water and extracted four times with200 ml. portions and three times with 100 ml. portions ofdichloroethylene. After drying overnight over 10 grams of MgSO thevolatiles were flashed off under reduced pressure, leaving 97 grams ofresidue that contained 82percent (vapor phase chromatographic analysis)of ethyl nitroacetate, for an overall yield of 60 percent based onnitromethane.

Preparation of Salt of Gramine and Ethyl Nitroacetate 7.2 grams (0.0444mole) of the ethyl nitroacetate prepared above and 8.7 grams (0.05 mole)of gramine were stirred together at room temperature in 100 C. toluenefor 1 hour. The precipitate was filtered off and dried in a vacuum atC., and 1 mm. Hg. 13.9 grams of product, m.p. 74-75 C., were obtained.Infrared and NMR analyses indicated that the product was the graminesalt of ethyl nitroacetate. A thin layer chromatographic analysis of thesalt showed only one spot, in-

Preparation of Tryptophan 7.7 grams (0.025 mole) of the gramine-ethylnitroacetate salt prepared above were dissolved in 100 ml. toluene. Thesolution was refluxed overnight under a stream of nitrogen. The offgases were passed through a scrubber containing 0.025 mole of aceticacid. 0.0248 mole, representing 99 percent of theory, of dimethyl aminewas trapped in the scrubber. A thin-layer chromatographic analysis ofthe reaction mixture showed only one spot. The solution was diluted withan additional 200 ml. of toluene and charged to an autoclave togetherwith 1.0 gram 5 percent palladium on carbon. Reduction was completeafter the mixture was stirred for 4.5 hours at 600 p.s.i. hydrogen andC. A thin-layer chromatographic analysis of the filtered solution showedone major spot corresponding to ethyl a-amino-B-(B-indole) propionatc.Dry HCl gas was passed through the chilled solution until the amine wascompletely precipitated. The precipitate was collected by filtration anddried. The product weighed 5.7 grams fora yield of percent. 2.69 grams(0.01 mole) of the product was dissolved in 8 ml. of 20 percent sodiumhydroxide and 25 ml. ethanol. After being stirred for 1 day at room tem'perature, the solution was acidified with glacial acetic acid to pH 5.9.The mixture was chilled for 2 hours at 0 C. and the product wasrecovered by filtration. There was thus obtained 1.62 grams oftryptophan, m.p. 276-278 C. Analysis indicated that an additional 0.28gram of tryptophan was present in the filtrate. The overall yield oftryptophan, including the tryptophan present in the filtrate, was thus79 percent based on gramine.

lclaim:

l. A process for preparing a gramine salt of an ester of nitroaceticacid having the formula wherein R is a lower alkyl, phenyl or loweralkyl substituted phenyl.

2. A gramine salt of an ester of nitroacetic acid having the formulawherein R is a lower alkyl, phenyl or lower alkyl substituted phenyl.